Anion Exchange Mechanisms in Bacteria

细菌中的阴离子交换机制

• 批准号: 9603997
• 负责人: Peter Maloney
• 金额: $ 36万
• 依托单位: Johns Hopkins University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-03-15 至 2000-02-29
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9603997&HistoricalAwards=false
• 关键词: Anion; Exchange; Mechanisms; Bacteria

9603997 Maloney This project concerns Oxlt, a novel transport protein found in the Gram-negative anaerobe, Oxalobacter formigenes. OxlT catalyzed the exchange of external divalent oxalate for internal monovalent formate. This electrogenic exchange, along with proton consumption during intracellular decarboxylation, constitutes an "indirect proton pump" that generates a proton-motive force allowing O. formigenes to extract energy from oxalate. It is now thought such protonmotive metabolic cycles are widely spread in microbiology, and to understand these events at a molecular level this project concentrates on studies on OxlT, the antiport protein at the center of this process in O. formigenes. Antibodies against the OxlT N-terminus are available, the gene is cloned, and functional protein is purified after expression in Escherichia coli. Therefore, tools are available to study structure-function relationships in four areas of progressively increasing resolution: (1) Initial studies will focus on studies of OxlT topology, using peptidespecific antibodies, gene fusions and SH-directed probes, guided by predictions derived from hydropathy analysis of the amino acid sequence. (2) Site-directed mutagenesis will then be used to generate a panel of single-cysteine derivatives to be probed with impermeant SH-active reagents in order to localize the OxlT translocation pathway. (3) Mutagenesis will also be used to identify functionally important residues, with emphasis on K355, which may be the lone charged residue in the OxlT hydrophobic core; K355 may form part of an anion binding center. (4) Concurrently, capitalizing on the stability of the OxlT/substrate complex and on recent success in protein purification, trials of 2-dimensional crystallization will be conducted. Together, this work will extend an understanding of OxlT from the level of cell physiology to that of molecular biology and biophysics. This project is about OxlT, a novel transport protein found in the soil bacterium, Ox alobacter formigenes. OxlT catalyzes the transmembrane exchange of two anions that differ in net charge, so that a membrane potential arises as one (divalent oxalate) moves inward while the other (monovalent formate) moves outward. Similarly, when inside the cell, oxalate is transformed into formate in a reaction that consumes a single proton. Overall, then, the metabolism of oxalate generates electrical and chemical (ph) gradients that comprise a "protonmotive force" which O. formigenes uses to power other membrane-associated events. Such proton-motive metabolic cycles appear to be widely spread in microorganisms. To understand these events at a molecular level this project concentrates on studies of OxlT, the antiport protein at the center of the process in O. formigenes. Initial studies will focus on the overall organization of OxlT in the cell membrane, using biochemical, genetic and immunological tools. Mutagenesis will then be used to change the molecule into one suitable for probing with reagents that may localize the substrate transport pathway and identify functionally important residues. Finally, trials of 2-dimensional crystallization will be conducted to capitalize on the stability of the OxlT-substrate complex. Together, such experiments should experiments should extend an understanding of OxlT from the level of cell physiology to that of molecular biology and biophysics.

9603997 Maloney 该项目涉及 Oxlt，一种在革兰氏阴性厌氧菌、产酸草杆菌中发现的新型转运蛋白。 OxlT 催化外部二价草酸盐交换为内部一价甲酸盐。 这种生电交换以及细胞内脱羧过程中的质子消耗构成了“间接质子泵”，它产生质子动力，使 O. formigenes 能够从草酸盐中提取能量。 现在人们认为这种质子动力代谢循环在微生物学中广泛传播，为了在分子水平上理解这些事件，该项目集中于对 OxlT 的研究，OxlT 是 O. formigenes 中这一过程的中心蛋白。 针对 OxlT N 末端的抗体已可用，基因已克隆，并且在大肠杆菌中表达后纯化了功能蛋白。 因此，有工具可用于研究分辨率逐渐提高的四个领域的结构-功能关系：（1）初步研究将集中于 OxlT 拓扑结构的研究，使用肽特异性抗体、基因融合和 SH 定向探针，以氨基酸序列水病分析的预测为指导。 (2) 然后使用定点诱变生成一组单半胱氨酸衍生物，用不渗透的 SH 活性试剂进行探测，以定位 OxlT 易位途径。 (3) 诱变还将用于鉴定功能上重要的残基，重点是 K355，它可能是 OxlT 疏水核心中唯一的带电残基； K355可以形成阴离子结合中心的一部分。 (4)同时，利用OxlT/底物复合物的稳定性和最近在蛋白质纯化方面的成功，将进行二维结晶的试验。 总之，这项工作将把对 OxlT 的理解从细胞生理学水平扩展到分子生物学和生物物理学水平。 该项目是关于 OxlT，一种在土壤细菌 Ox alobacter formigenes 中发现的新型转运蛋白。 OxlT 催化两种净电荷不同的阴离子的跨膜交换，因此当一种阴离子（二价草酸根）向内移动而另一种阴离子（一价甲酸根）向外移动时，就会产生膜电位。 同样，在细胞内部，草酸盐在消耗单个质子的反应中转化为甲酸。 总体而言，草酸盐的代谢产生电和化学 (ph) 梯度，其中包括“质子动力”，O. formigenes 使用该力为其他膜相关事件提供动力。 这种质子动力代谢循环似乎在微生物中广泛传播。 为了在分子水平上了解这些事件，该项目集中于 OxlT 的研究，OxlT 是 O. formigenes 过程的中心蛋白。 初步研究将集中于细胞膜中 OxlT 的整体组织，使用生化、遗传和免疫学工具。 然后使用诱变将分子改变为适合用试剂探测的分子，这些试剂可以定位底物转运途径并识别功能上重要的残基。 最后，将进行二维结晶试验，以利用 OxlT-底物复合物的稳定性。 总之，这些实验应该将 OxlT 的理解从细胞生理学水平扩展到分子生物学和生物物理学水平。